A PIP2-stabilized syntaxin-1a structure mapped with transition metal ion FRET and unnatural fluorescent amino acids at the plasma membrane

Ca²⁺-triggered exocytosis from neurons and endocrine cells is regulated by neuronal soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins. Conformational changes in syntaxin-1-the plasma membrane t-SNARE-are essential for vesicle docking and exocytosis. The nature of these conformational changes on the plasma membrane in living cells, however, remains largely unknown. Here, we develop a fluorescence system to map short-range conformational changes in syntaxin-1a in native plasma membranes of unroofed cells. We use a fluorescence resonance energy transfer (FRET) technique that employs site-specific protein labeling with unnatural fluorescent amino acids as donor fluorophores and colored transition metal ion acceptors bound to engineered di-histidine sites to map angstrom-scale distances. We find that phosphatidylinositol 4,5-bisphosphate (PIP2) regulates a conformational change in syntaxin-1a by modulating the structure of syntaxin-1a and its interaction with Munc18-1. Our results uncover new regulatory mechanisms of syntaxin-1a by PIP2 in the steps leading to Ca²⁺-triggered exocytosis.